Part:BBa_K4209000
tal-fjo, a gene encoding for a tyrosine amino lyase that converts Tyr to pHCA
This basic part is a gene encoding for a tyrosine amino lyase that allows the conversion of tyrosine to p-coumaric acid (pHCA). This part works in combination with SULT1A1 (BBa K4209001) for the production of zosteric acid, our product of interest. The cloned composite parts are BBa_K4209007 and BBa_K4209008.
Introduction
Tal-fjo is a gene encoding for a tyrosine amino lyase (from Flavobacterium johnsoniae), which transforms L-tyrosine in p-coumaric acid. The p-coumaric acid will then be transformed in zosteric acid, our product of interest, thanks to the sulfotransferase encoded by SULT1A1. For this reaction to occur the cell needs to transport sulfate into the cells by genes encoding for transporters (cysP (from Bacillus subtillis) or cysPUWA (from E.coli).
Zosteric acid (ZA) is a bioactive molecule found in Zostera marina eelgrass (Taokaew et al. 2012) that, if bound to a surface, can deter the attachment of marine microorganisms and bryozoans. This compound has already been studied and classified as an effective ecologically sound anti-biofilm against quagga mussels (Taokaew et al. 2012), making it a very attractive candidate to be considered a major part in our project.
The metabolic pathway is visualized in figure 1 and the genes needed are explained in table 1. The metabolic pathway for ZA production has therefore already been explored and cloned in E.coli, but its production has only been assessed in 950 µL of medium. We will therefore clone the genes in plasmids different from the ones used in literature and try to swap the position of the genes relative to the inducible promoter to see if some of our constructs will produce more ZA than others. Bacteria will be grown in 200 ml flasks instead of 96 well plates as found in literature (Ram et al. 2012).
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Figure 1: Metabolic pathway for ZA production. (Adapted from Jendresen et al. 2019)
Design
For our project, we wished to produce ZA in large quantities using engineered bacterial cells as “cell factories” to assess its efficacy as an anti-foulant agent against quagga mussels. To be able to produce zosteric acid, we engineer plasmids containing SULT1A1 and TAL in different orders: one with SULT1A1 and then TAL and in the other direction. SULT1A1 and TAL were placed in a pET17b plasmid with an IPTG inducible promoter for protein expression in E.coli (see figure 2). The T7 promoter is activated once we add IPTG to the growth media, which switches on the expression of the genes.
SULT1A1 and TAL have been synthesized. We cloned the two plasmids individually in E. coli DH5α strain, then extracted them using a plasmid purification kit. We co-transformed E. coli BL21 DE3 cells with a plasmid construct containing SULT1A1 and TAL and a plasmid construct containing genes responsible for the sulfate intake to allow the production of ZA.
Successful co-transformants were isolated by plating cells on selective media supplemented with both the antibiotics kanamycin and ampicillin. The ZA produced was then released in the extracellular space and diluted in the media. We decided to use M9 as growing media for our transformed cells as it is a minimal media and contains low quantities of sugars and salts, making it easier to detect ZA. Detection of ZA was performed with high performance liquid chromatography (HPLC), as the sulfate group in the ZA made it undetectable using the GC-MS available in our department. Upon confirmation of the production of ZA, we have planned to concentrate and purify it using distillation. This would allow us to create either a liquid solution containing a high concentration of ZA or even a powder containing ZA and salts.
Results
First, we cloned tal-fjo and SULT1A1 genes into the pET17b plasmid backbone.
The tal-fjo and SULT1A1 genes necessary for the catalysis of the two conversion reactions (tyrosine to pHCA, and pHCA to ZA, respectively) were ordered from Twist Bioscience.
We successfully PCR-amplified all the genes and linear backbone fragments required to perform our cloning (Figure 3). Then we cloned the genes into their respective backbone through Gibson assembly and transformed the cloned plasmid into E. coli DH5α. By performing colony PCR and subsequent Sanger sequencing on the resulting transformants, we also were able to confirm the successful cloning of all of our plasmids.
The newly-built plasmids were then co-transformed into E. coli BL21 (DE3), a strain more adapted for protein production. Indeed, we wished to generate four distinct strains each containing different combination of two plasmids, one bearing sulphate uptake genes and one bearing the the catalytic enzymes. Unfortunately, we were not able to co-transform E. coli BL21 (DE3) containing pCola_Duet_P_DNCQ and pET_17b_Tal_SULT1A1. We, however, successfully obtained the three following co-transformant that we analyzed further for ZA production.
Zosteric acid production from our engineered strains
To test the ability of our engineered strains to produce ZA, we used HPLC to analyse the supernatant of our cells grown in liquid cultures for the detection of ZA and pHCA.
a.Expected results from HPLC:
We first purchased the pure compounds ZA and pHCA and ran them into the HPLC as standards (Fig 5). This allowed us to confirm we could detect ZA and pHCA with our method, with a retention time (RT) of 5 and 9 min, respectively.
Next, we analysed the supernatants of our three mutant strains to assess ZA production To do so, we cultured overnight the strains in M9 medium supplemented with 4 mM of pHCA,10 mM of K2SO4-- and 0.1 mM IPTG induction. When supplied with all molecules, we expected our engineered cells to utilise pHCA and produce ZA, resulting in an absorbance peak at 5 min RT corresponding to ZA and the reduction of the peak at 9 min RT for pHCA, with respect to a non-induced control (Fig.6).
Indeed, as a reference control we additionally analysed the supernatant of cells grown with the substrates pHCA and K2SO4- (Figure 6) but without IPTG induction.In these conditions, we expected to observe no production of ZA but a significant amount of pHCA corresponding to the added initial concentration that cannot be utilised by the cells.
Lastly, we prepared a second control for each mutant strain where we added IPTG, but no supplementation of pHCA and K2SO4-. The rationale of this control was to assess the efficacy of the tal-fjo gene-encoded protein to effectively convert tyrosine into pHCA. Without added sulphate and pHCA, we indeed reasoned that any detected pHCA would be the product of the tal-fjo protein activity. pHCA would also most likely accumulate as no added sulphate would limit its further conversion to ZA. Here, we therefore expected to observe production of pHCA and virtually no ZA. In the case where the basal level of sulphate present in the M9 media used for our experiment would be sufficient for conversion of ZA, we would then expect minimal production of ZA and reduced amount of pHCA (Figure 6).
b.Observed HPLC results:
After a few preliminary analyses to determine the best conditions under which to perform the HPLC, the following results were obtained:
Indeed, we analysed the supernatant of our three different strains, each bearing a different combination of the transport and catalytic plasmids. We, however, observed the strongest signal with the P-ST and PUWA-ST co-transformants. For the sake of clarity, we, therefore, decided to report here only the results obtained for these strains (Fig. 7).
The absorption profile of the supernatant isolated from the PUWA-ST strain interestingly showed that in the control 2 growth conditions, cells were able to convert tyrosine into pHCA as a remarkable peak with the correct retention time can be observed while the molecule was not added here (Fig. 7C, PUWA-ST-CHEM). However, comparing the test and control 1 conditions for this strain, no decrease of pHCA and detection of ZA can be seen, which suggests that no pHCA is being utilised. Altogether, these observations indicate that the tal-fjo is functional in producing pHCA but the PUWA does not appear to allow significant uptake of sulphate to permit conversion of pHCA to ZA.
On the other hand, the P-ST strain showed no detectable pHCA in the control 2 conditions, while we observed a clear decrease in pHCA between the test and control 1 conditions when it was added in. Although we do not detect a peak for ZA itself, we argue that this data suggests that ZA is being produced, albeit below the detection limit of the method used, as pHCA decreases specifically when the cells are induced. We thus considered a notable decrease of pHCA as a proxy for ZA production. Comparing our two strains, we can also conclude that the cysP protein seems more functional than the cysPUWA protein in sulphate uptake which is effectively used to convert pHCA to ZA, as the decrease of pHCA could only be detected with the cysP gene.
In conclusion, we are pleased to report that our data so far suggest our P-ST strain is able to convert pHCA to ZA. Further work will, however, be carried out to improve our growth conditions and the detection of ZA in order to confirm this.
Future perspective
The testing on mussels was planned with known concentrations of ZA: 500 ppm, 1000 ppm and 2000 ppm. 100 ml of lake water with the right concentration of ZA will be placed into a box with 4 quagga mussels, as we noticed along the way that having multiple mussels per box increases the probability of attachment. The attachment of each mussel will be tested at regular intervals of 3 hours: 9 am, 12 am, 3 pm and 6 pm. Data will then be tabulated and subjected to an appropriate statistical test to understand if ZA plays a role in attachment probability.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1303
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1303
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1303
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1303
Illegal AgeI site found at 925
Illegal AgeI site found at 1267
Illegal AgeI site found at 1357 - 1000COMPATIBLE WITH RFC[1000]
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